Loss of chromosomal stability is a common feature of human carcinomas. How cells maintain chromosomal integrity is incompletely understood; however it is clear that many pathways and protein complexes are involved. Maintenance of the correct chromosomal content (euploidy) is an important contributor to overall chromosomal stability. Emerging data indicate that epigenetic silencing of pericentromeric repetitive DNA elements plays a key role in maintenance of euploidy. Defective pericentromeric silencing is sufficient to induce chromosomal instability and, in animal models, malignancy. This application focuses on novel functions for the sirtuin SIRT6 in tumor suppression, pericentromeric silencing, and ploidy maintenance. The sirtuins are a family of protein deacetylases that promote many aspects of healthspan in mammals, among them tumor suppression. The long- term goal of the laboratory is to elucidate mechanisms by which sirtuins, in particular SIRT6, promote healthspan. Recently published work by the applicant's laboratory has shown that SIRT6 functions as an intestinal tumor suppressor in humans and mice, at least in part via attenuation of MYC and HIF1 signaling. The objective of this application is to elucidate a third role of SIRT6 in tumor suppression, specifically in pericentromeric silencing and chromosomal stability. The central hypothesis of this application is that SIRT6 promotes epigenetic silencing of pericentromeric DNA to promote euploidy maintenance, a function that contributes to its tumor suppressor function. The rationale for these studies is that a mechanistic dissection of the relationships between SIRT6, pericentromeric silencing, and euploidy will contribute to an enhanced understanding of chromosomal stability overall. This work will be carried out in three Specific Aims. First, the significance of SIRT6's interactions with known chromatin silencing factors will be elucidated. Potential mitotic defects in SIRT6-deficient cells will be identified b live-cell imaging. Second, the impact of loss of SIRT6 function will be assessed in intestinal epithelial cells and adenomas with respect to malignant properties and induction of aneuploidy. Third, the ability of SIRT6 overexpression to suppress adenomatosis will be tested in vivo. The approach is innovative, in that the contribution of pericentromeric silencing to ploidy maintenance is still incompletely understood, and SIRT6 has not previously been linked to this process. The work is significant, in that it is likely to provide new insights into mechanisms of ploidy maintenance, which in turn is a major factor in overall genomic stability and tumor suppression.